Epithelial tubes form the basis of numerous organ systems, including the vertebrate renal (kidney) and circulatory systems. During the normal course of development, tubes form from non-epithelial cells which polarize to generate different apical and basal surfaces, and must also form and maintain junctions either with neighboring cells or with themselves. Although a number of junction complexes have been identified, including the cadherin/catenin complex and the discs-large complex, little is known about how epithelial junctions are initially assembled and then maintained during tubulogenesis. The simplest tubes are unicellular in nature, and may present the most reductive models for junction formation and maintenance. We propose to study junction formation in the unicellular tubes found in the excretory system of the C. elegans. In a recent screen for genes required during excretory system development or maintenance was recently performed in our lab, we found a mutation in the daj-1 gene. The daj-1 gene encodes 14 extracellular Leucine Rich Repeats, a common protein- protein binding motif. Preliminary analysis shows that daj-1 is required for maintenance of the autocellular junction of the excretory pore cell in C. elegans. We propose the elucidation of the role of daj-1 in junction formation or maintenance in the excretory system, and the determination of the relationships between proteins with extracellular LRR motifs in C. elegans. In Aim 1 we will determine the requirements for daj-1 in the excretory system. We will first elucidate the full phenotype of daj-1 mutants using live imaging of junctional markers. To determine where and when DAJ-1 is expressed we will create fluorescent-tagged DAJ-1 reporters and follow them by live imaging. Finally, we will determine where and when daj-1 expression is required by rescuing daj-1(-) zygotic lethality in specific tissues, and by temporally rescuing daj-1(-) zygotic lethality in specific tissues by induction of a heat-shock promoter. In Aim 2 we will determine how DAJ-1 carries out its function in the excretory system. We will first perform structure/function analyses to determine which domains are critical to DAJ-1 activity in the excretory system. We will then investigate whether DAJ-1 and another related eLRR excretory protein LET-4 have distinct functions in the excretory system. Finally we will determine which proteins DAJ-1 interacts with in order to carry out its function in the excretory system using biochemical techniques (co-immunoprecipitation, pull-down).